Replacing the Traditional Colony-Forming
Cell Assay with the HALO® Platform
or
How Total Colony Counts can be Converted to ATP Equivalent Concentrations to Standardize the Colony-Forming Cell Assay
7 Day HALO® Platform acknowledged by Stem Cell Technologies
In a surprising and dramatic turn of events, Stem Cell Technologies has reversed its course and now claims that counting undifferentiated colonies at 7 days derived from human cells, is the same as counting differentiated colonies at 14 days. This was presented at the International Society of Cellular Therapy meeting in San Diego, CA on May 4th, 2008 as a poster and on May 6th, 2008 as a sponsored corporate workshop.
This confirms one of the basic concepts of the HemoGenix® HALO® assay first published in 2003 (Current Opinion in Drug Discovery & Development 6:100-109) and demonstrated on this page (please scroll down).
It has taken Stem Cell Technologies 6 years to become "enlightened" and confirm one of the basic concepts of the HALO® Platform:
In the traditional colony-forming assay, the cultures are left to incubate so that the cells within the colonies differentiate. This allows different types of colonies to be identified. However, in this assay, whole colonies are counted as shown in the picture below.
When cells are incubated in methyl cellulose, small clusters are initially formed. These clusters consist of undifferentiated, proliferating cells, which we designate "proliferation units" (PUs). These "proliferation units" are also present in mature colonies. Rather than counting a single whole colony, the "proliferation units" are the clusters that make up the whole colonies as shown in the pictures below.
Although more difficult and time-consuming to count, it is these clusters or "proliferation units" that correlate with the luminescence readout. The diagrams below show the growth kinetics of clusters or "proliferation units" and how they correlate with the ATP luminescence readout.
Time Course of Colony Formation: Correlation between "Proliferation Units" and bioluminescence as a measure of proliferation.
TIME COURSE BETWEEN "PROLIFERATION UNITS" AND LUMINESCENCE FOR HUMAN CFC-GEMM, BFU-E AND GM-CFC
(Published in Current Opinion in Drug Discovery & Development, 2003, 6:100-109)
TIME COURSE BETWEEN "PROLIFERATION UNITS" AND LUMINESCENCE FOR HUMAN G-CFC, M-CFC, Mk-CFC, T-CFC AND B-CFC.
(Published in Current Opinion in Drug Discovery & Development, 2003, 6:100-109)
TIME COURSE BETWEEN "PROLIFERATION UNITS" AND LUMINESCENCE FOR MOUSE CFC-GEMM, BFU-E, GM-CFC AND Mk-CFC.
(Published in Current Opinion in Drug Discovery & Development, 2003, 6:100-109)
When HALO®-96 MeC is performed, the ATP measured by bioluminescence as a function of proliferation for human cultured is measured at 7 days, instead of at 14 days, as is the case for the CFC assay. At 7 days, proliferation is exponential, but differentiation is either low or has not begun. Similarly, for mouse cells, instead of waiting 7 days using the CFC assay, HALO® only requires 4 days.
HALO® Performed at 7 Days Predicts Colony Counts at days 10 and 14.
When the Relative Luminescence Units measured on day 7 of incubation for human bone marrow CFC-GEMM are plotted against the number of "proliferation units" manually counted on days 10 and 14 as a function of cell dose the correlations shown bellow are obtained. This graph demonstrates that luminescence measured on day 7 can predict the manual enumeration of "proliferation units" on days 10 or day 14.
(Published in Current Opinion in Drug Discovery & Development, 2003, 6:100-109)
FURTHER CORRELATION OF HALO® FORMATS WITH THE COLONY-FORMING ASSAY.
Replacement of the Traditional Colony-Forming Assay with HALO®
The following 3-dimensional graph shows a direct relationship between the plated cell concentration, the total number of colonies counted and the results from the HALO®-96 MeC Platform for human bone marrow CFC-GEMM.
The next 3-dimensional graph shows a similar relationship between the cell dose, HALO®-96 MeC and HALO®-96 SEC.
If a relationship exists between cell dose and the colony-forming assay and either the HALO®-96 MeC or HALO®-96 SEC, then it would be expected that a direct relationship exists between the colony-forming assay and both HALO® Platforms. The graph below shows this to be true.
CONCLUSION
These results clearly demonstrate that a correlation exists between the traditional colony-forming assay and the HALO®-96 MeC and HALO®-96 SEC Platforms.
If the colony-forming assay is performed under exactly the same conditions as HALO®, total colony counts can be expressed in ATP (μM) production equivalents. Only in this manner can the colony-forming assay be standardized. This is the basis of the first and only standardized colony-forming stem and progenitor cell potency assay, CAMEO™-96.
Therefore, both HALO® Platforms are an alternative to the colony-forming assay and can replace it completely for the majority of applications.